Project Summary/Abstract Bacteriophages facilitate the evolution of bacterial pathogenicity by imposing selection for resistance to infection and by horizontal gene transfer of host genes to new bacteria. Temperate phages also often carry toxins and factors that convert benign bacteria into virulent pathogens, and they promote the spread of infection. In order to infect a host bacterium, the phage must adsorb to a cell surface, and then breach the integrity of the cell envelope. Most phages utilize their tails to initiate infection but the short-tailed Podoviridae cannot directly access the cell cytoplasm in order to deliver their genome. The initial steps of infection leading to genome ejection, especially how the channel, extending from the phage head through the cell membrane(s) and into the cell cytoplasm forms, remain the least understood aspects of any phage life cycle. This channel is essential for genome ejection. Some phages, salmonellaphage P22 among them, carry proteins inside their head that are ejected into cells to functionally extend tail length and create a trans-envelope channel for DNA transport. Our central hypothesis is that after adsorption, the P22 tail ejection nanomachine initiates a cascade of coordinated conformational changes in the virion that triggers ejection of three head proteins, resulting in a trans-envelope channel for genome transport into the host bacterium. Our immediate objective is to document these conformational rearrangements by characterizing structural intermediates during infection, localizing the ejected proteins in the infected cell. Using molecular genetics of both phage and host, and in combination with high throughput and high-resolution cryo- electron tomography (cryoET), we will analyze abortive infections by defective virions. Our novel and truly cross-disciplinary approach has already revealed essential structural intermediates in the process of viral infection. Biochemical analyses of the three ejected proteins will provide complementary information on the size, structure and assembly of the trans-envelope channel. This study is already providing new molecular insights into the mechanistic pathways leading to phage infection.